Formation of the b subunit dimer is necessary for interaction with F1-ATPase.
In earlier work, we [McCormick, K. A., et al. (1993) J. Biol. Chem. 268, 24683-24691] observed that mutations at Ala-79 of the b subunit affect assembly of F1F0 ATP synthase. Polypeptides modeled on the soluble portion of the b subunit (bsol) with substitutions at the position corresponding to Ala-79 have been used to investigate secondary structure and dimerization of the b subunit. Circular dichroism spectra and chymotrypsin digestion experiments suggested that the recombinant polypeptides with Ala-79 substitutions assumed conformations similar to the bsol polypeptide. However, cross-linking studies of the Ala-79 substitution bsol polypeptides revealed defects in dimerization. The efficiency of dimer formation appeared to be related to the capacity of the altered bsol polypeptides for competing with F1-ATPase for binding to F1-depleted membrane vesicles. Ala-79 substitution polypeptides displaying limited dimerization, such as bsol Ala-79-->Leu, were shown to elute with F1-ATPase during size exclusion chromatography, suggesting a specific interaction. Sedimentation equilibrium studies indicated that 8% of the bsol Ala-79-->Leu polypeptide was in the form of a 30.6 kDa dimer and 92% a 15.3 kDa monomer. When the dimer concentration of bsol Ala-79-->Leu was normalized to the concentration of bsol, both had virtually identical capacities for competing with F1-depleted membrane vesicles for binding F1-ATPase. The result indicated that the amount of dimer formed is directly proportional to its ability to bind F1-ATPase. This suggests that formation of the b subunit dimer may be a necessary step preceding F1-ATPase binding in the assembly of the enzyme complex.
Sorgen
P L
PL
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610, USA.
Bubb
M R
MR
McCormick
K A
KA
Edison
A S
AS
Cain
B D
BD
D019943:Amino Acid Substitution / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry
D010455:Peptides; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases